Divinyl ether fatty acid synthesis in late blight-diseased potato leaves.
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We conducted a study of the patterns and dynamics of oxidized fatty acid derivatives (oxylipins) in potato leaves infected with the late-blight pathogen Phytophthora infestans. Two 18-carbon divinyl ether fatty acids, colneleic acid and colnelenic acid, accumulated during disease development. To date, there are no reports that such compounds have been detected in higher plants. The divinyl ether fatty acids accumulate more rapidly in potato cultivar Matilda (a cultivar with increased resistance to late blight) than in cultivar Bintje, a susceptible cultivar. Colnelenic acid reached levels of up to approximately 24 nmol (7 microgram) per g fresh weight of tissue in infected leaves. By contrast, levels of members of the jasmonic acid family did not change significantly during pathogenesis. The divinyl ethers also accumulated during the incompatible interaction of tobacco with tobacco mosaic virus. Colneleic and colnelenic acids were found to be inhibitory to P. infestans, suggesting a function in plant defense for divinyl ethers, which are unstable compounds rarely encountered in biological systems. (+info)
Induction of monocyte binding to endothelial cells by MM-LDL: role of lipoxygenase metabolites.
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Treatment of human aortic endothelial cells (EC) with minimally oxidized LDL (or minimally modified LDL, MM-LDL) produces a specific pattern of endothelial cell activation distinct from that produced by LPS, tumor necrosis factor-alpha, and interleukin-1, but similar to other agents that elevate cAMP. The current studies focus on the signal transduction pathways by which MM-LDL activates EC to bind monocytes. We now demonstrate that, in addition to an elevation of cAMP, lipoxygenase products are necessary for the MM-LDL response. Treatment of EC with inhibitors of the lipoxygenase pathway, 5,8,11, 14-eicosatetraynoic acid (ETYA) or cinnamyl-3, 4-dihydroxy-alpha-cyanocinnamate (CDC), blocked monocyte binding in MM-LDL-treated EC (MM-LDL=118+/-13%; MM-LDL+ETYA=33+/-4%; MM-LDL+CDC=23+/-4% increase in monocyte binding) without reducing cAMP levels. To further investigate the role of the lipoxygenase pathway, cellular phospholipids were labeled with arachidonic acid. Treatment of cells for 4 hours with 50 to 100 microg/mL MM-LDL, but not native LDL, caused a 60% increase in arachidonate release into the medium and increased the intracellular formation of 12(S)-HETE (approximately 100% increase). There was little 15(S)-HETE present, and no increase in its levels was observed. We demonstrated that 12(S)-HETE reversed the inhibitory effect of CDC. We also observed a 70% increase in the formation of 11,12-epoxyeicosatrienoic acid (11, 12-EET) in cells treated with MM-LDL. To determine the mechanism of arachidonate release induced by MM-LDL, we examined the effects of MM-LDL on intracellular calcium levels. Treatment of EC with both native LDL and MM-LDL caused a rapid release of intracellular calcium from internal stores. However, several pieces of evidence suggest that calcium release alone does not explain the increased arachidonate release in MM-LDL-treated cells. The present studies suggest that products of 12-lipoxygenase play an important role in MM-LDL action on the induction of monocyte binding to EC. (+info)
Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis.
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Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place. (+info)
Formation of lipoxygenase-pathway-derived aldehydes in barley leaves upon methyl jasmonate treatment.
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In barley leaves, the application of jasmonates leads to dramatic alterations of gene expression. Among the up-regulated gene products lipoxygenases occur abundantly. Here, at least four of them were identified as 13-lipoxygenases exhibiting acidic pH optima between pH 5.0 and 6.5. (13S,9Z,11E,15Z)-13-hydroxy-9,11,15-octadecatrienoic acid was found to be the main endogenous lipoxygenase-derived polyenoic fatty acid derivative indicating 13-lipoxygenase activity in vivo. Moreover, upon methyl jasmonate treatment > 78% of the fatty acid hydroperoxides are metabolized by hydroperoxide lyase activity resulting in the endogenous occurrence of volatile aldehydes. (2E)-4-Hydroxy-2-hexenal, hexanal and (3Z)- plus (2E)-hexenal were identified as 2,4-dinitro-phenylhydrazones using HPLC and identification was confirmed by GC/MS analysis. This is the first proof that (2E)-4-hydroxy-2-hexenal is formed in plants under physiological conditions. Quantification of (2E)-4-hydroxy-2-hexenal, hexanal and hexenals upon methyl jasmonate treatment of barley leaf segments revealed that hexenals were the major aldehydes peaking at 24 h after methyl jasmonate treatment. Their endogenous content increased from 1.6 nmol.g-1 fresh weight to 45 nmol.g-1 fresh weight in methyl-jasmonate-treated leaf segments, whereas (2E)-4-hydroxy-2-hexenal, peaking at 48 h of methyl jasmonate treatment increased from 9 to 15 nmol.g-1 fresh weight. Similar to the hexenals, hexanal reached its maximal amount 24 h after methyl jasmonate treatment, but increased from 0.6 to 3.0 nmol.g-1 fresh weight. In addition to the classical leaf aldehydes, (2E)-4-hydroxy-2-hexenal was detected, thereby raising the question of whether it functions in the degradation of chloroplast membrane constituents, which takes place after methyl jasmonate treatment. (+info)
Cucumber cotyledon lipoxygenase during postgerminative growth. Its expression and action on lipid bodies.
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In cucumber (Cucumis sativus), high lipoxygenase-1 (LOX-1) activity has been detected in the soluble fraction prepared from cotyledons of germinating seeds, and the involvement of this enzyme in lipid turnover has been suggested (K. Matsui, M. Irie, T. Kajiwara, A. Hatanaka [1992] Plant Sci 85: 23-32; I. Fuessner, C. Wasternack, H. Kindl, H. Kuhn [1995] Proc Natl Acad Sci USA 92: 11849-11853). In this study we have investigated the expression of the gene lox-1, corresponding to the LOX-1 enzyme. LOX-1 expression is highly coordinated with that of a typical glyoxysomal enzyme, isocitrate lyase, during the postgerminative stage of cotyledon development. In contrast, although icl transcripts accumulated in tissue during in vitro senescence, no accumulation of lox-1 mRNA could be observed, suggesting that lox-1 plays a specialized role in fat mobilization. LOX-1 is also known to be a major lipid body protein. The partial peptide sequences of purified LOX-1 and lipid body LOX-1 entirely coincided with that deduced from the lox-1 cDNA sequence. The data strongly suggest that LOX-1 and lipid body LOX-1 are derived from a single gene and that LOX-1 can exist both in the cytosol and on the lipid bodies. We constructed an in vitro oxygenation system to address the mechanism of this dual localization and to investigate the action of LOX-1 on lipids in the lipid bodies. LOX-1 cannot act on the lipids in intact lipid bodies, although degradation of lipid body proteins, either during seedling growth or by treatment with trypsin, allows lipid bodies to become susceptible to LOX-1. We discuss the role of LOX-1 in fat mobilization and its mechanism of action. (+info)
Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity.
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The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 +/- 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 +/- 4 and 65 +/- 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose-response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were "capped" with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB "cap" after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity. (+info)
The diversity of the lipoxygenase family. Many sequence data but little information on biological significance.
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Lipoxygenases form a family of lipid peroxidising enzymes, which oxygenate free and esterified polyenoic fatty acids to the corresponding hydroperoxy derivatives. They are widely distributed in both the plant and animal kingdoms. During the last couple of years more and more lipoxygenase isoforms have been discovered but for most of them the biological significance remains unclear. This review attempts to classify the currently known mammalian lipoxygenase isoforms and critically reviews the concepts for their biological importance. (+info)
When and why a water-soluble antioxidant becomes pro-oxidant during copper-induced low-density lipoprotein oxidation: a study using uric acid.
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The inclusion of uric acid in the incubation medium during copper-induced low-density lipoprotein (LDL) oxidation exerted either an antioxidant or pro-oxidant effect. The pro-oxidant effect, as mirrored by an enhanced formation of conjugated dienes, lipid peroxides, thiobarbituric acid-reactive substances and increase in negative charge, occurred when uric acid was added late during the inhibitory or lag phase and during the subsequent extensive propagation phase of copper-stimulated LDL oxidation. The pro-oxidant effect of uric acid was specific for copper-induced LDL oxidation and required the presence of copper as either Cu(I) or Cu(II). In addition, it became much more evident when the copper to LDL molar ratio was below a threshold value of approx. 50. In native LDL, the shift between the antioxidant and the pro-oxidant activities was related to the availability of lipid hydroperoxides formed during the early phases of copper-promoted LDL oxidation. The artificial enrichment of isolated LDL with alpha-tocopherol delayed the onset of the pro-oxidant activity of uric acid and also decreased the rate of stimulated lipid peroxidation. However, previous depletion of alpha-tocopherol was not a prerequisite for unmasking the pro-oxidant activity of uric acid, since this became apparent even when alpha-tocopherol was still present in significant amounts (more than 50% of the original values) in LDL. These results suggest, irrespective of the levels of endogenous alpha-tocopherol, that uric acid may enhance LDL oxidation by reducing Cu(II) to Cu(I), thus making more Cu(I) available for subsequent radical decomposition of lipid peroxides and propagation reactions. (+info)